This proposal addresses the molecular control of biological clocks in individual organisms and single cells. Higher plants comprise one system for these studies, in part because these organisms provide cis-acting DNA elements that mediate clock-controlled gene expression. To exploit this property, a non-invasive assay has been developed for clock-regulated gene expression in intact whole plants: the promoter of a clock-regulated gene (cab2) from Arabidopsis has been fused to a cDNA encoding firefly luciferase and transgenic plants were generated containing this construct. Preliminary data demonstrate that when plants transformed by the cab2- luciferase gene fusion are sprayed with luciferin, the plants f"glow" rhythmically, indicating that circadian-regulated transcription can be monitored in intact tissue. We propose to extend this technology to the analysis of dock-regulated transcription in single plant cells. The hypothesis is that individual cells of metazoan organisms can contain circadian clocks. The plant system will provide a powerful cell-level assay to address this issue. We will also identify biochemically the signal transduction pathways between the oscillator and the cab gene promoter in a plant suspension cell culture, to test the hypothesis that calmodulin-dependent pathways are important in regulating cab gene transcription. In another system, Drosophila, cis-acting elements of a 'clock locus' called period have been shown to be involved in mediating cyclic variations in per gene product levels. It is proposed to apply the luciferase reporter system to analysis of the molecular rhythm at the whole-fly level. The specific experiments will involve per-luciferase fusions and transgenic strains analogous to those in Arabidopsis. If individual flies can be shown to glow rhythmically, this will permit several important questions to be asked: Are per-related molecular cyclings actually correlated with behavioral circadian rhythms of Drosophila? The latter are controlled (in part) by per's action, and the flies rest/activity cycles persist for many days in constant conditions: so how long does the molecular rhythm persist? Can light-induced phase shifts of Drosophila's biological rhythmicity be shown to correlate with phase alterations of per-controlled protein cyclings? It is further suggested that the per-luciferase fusion system could eventually be applied to experiments aimed at asking at the molecular level if individual animal cells contain circadian clocks and if these pacemakers share common properties with those of plants? Given the ubiquity of circadian-regulated physiology, the identification of common clock components will have an impact on understanding the pacemaker mechanism and malfunctions associated with known features of human well-being.